Expression vectors, transfection systems, and method of use thereof

ABSTRACT

Expression vectors and transfection systems providing high expression of a desired polypeptide are provided. Also provided are methods of using the expression vectors and transfection systems and mammalian cells modified by these compositions and methods.

CROSS-REFERENCE TO RELATED APPLICATION

This application is related to provisional patent application Ser. No.60/162,930, filed Nov. 1, 1999, from which priority is claimed under 35USC §119(e)(1) and which application is incorporated herein by referencein its entirety.

FIELD OF THE INVENTION

The invention provides novel expression vectors that allow stable,high-level expression of a polypeptide of interest in a host cell,particularly mammalian cells. The invention also includes a transfectionsystem for mammalian cells using the constructs described herein. Theinvention described herein provides an efficient mechanism by which anydesired polypeptide can be expressed at high levels using novel celllines generated as described herein.

BACKGROUND OF THE INVENTION

Vectors based on lytic viruses such as polyoma have been used forshort-term expression, but tend to be unstable, and replicate many timesper cell cycle (Lebkowski, J. S., et al., MOL. CELL. BIOL. 4:1951-1960,1984). Vectors based on bovine papilloma virus have also been developedbut do not consistently replicate once per cell cycle (Gilbert, D. M.,et al., CELL 50:59-68, 1987; Ravnan, J.-B., et al., J. VIROL.66:6946-6952, 1992). Further bovine papilloma virus-based vectors show ahigh frequency of rearrangements (Ashman, C. R., et al., SOMATIC CELLMOL. GENET. 11:499-504, 1985; DuBridge, R. B., et al., MOL. CELL. BIOL.7:379-387, 1987).

In human and primate cells, vectors based on Epstein-Barr virus (EBV)have been developed (Yates, J., et al., PROC. NATL. ACAD. Sci. USA.81:3806-3810, 1984; Reisman, D., et al., MOL. CELL. BIOL. 5:1822-1832,1985; Lupton, S., et al., MOL. CELL. BIOL. 5:2533-2542, 1985). Thesevectors typically replicate once per cell cycle (Adams, A., J. VIROL.61:1743-1746, 1987; Yates, J. L., et al., J. VIROLOGY 65:483-488, 1991;Haase, S. B., et al., NUC. ACIDS RES. 19:5053-5058, 1991) and are stablymaintained over the long-term with a low mutation frequency (DuBridge,R. B., et al., MOL. CELL. BIOL. 7:379-387, 1987; DuBridge, R. B., etal., MUTAGENESIS 3:1-9, 1988; Drinkwater, N. R., et al., PROC. NATL.ACAD. SCI. USA 83: 3402-3406, 1986). These vectors have been used forcloning and expression studies in human and simian cells (Margolskee, R.F., et al., MOL. CELL. BIOL. 8:2837-2847, 1988; Young, J. M., et al.,GENE 62:171-185, 1988; Belt, P. B. G. M., et al., GENE 84:407-417, 1989;Peterson, C., et al., GENE 107:279-284, 1991). Stable transformationfrequencies are high because integration into the genome is notrequired, and recovery of cloned sequences is achieved by plasmidextraction. However, rodent cells are not permissive for EBVreplication, and no rodent counterpart of EBV has been described (Yates,J. L., et al., NATURE (LONDON) 313:812-815, 1985).

U.S. Pat. No. 4,959,317 (Sauer, et al.) discloses the use of Cre-Loxsite-specific recombination to achieve gene transfer in eukaryoticcells. However, the system described does not provide efficient orstable integration of transferred DNA into the host genome (see e.g.,(Sauer, et al., (1993) Methods in Enzymology 225: page 898). This islargely due to the fact that excision of transferred DNA out of thegenome, by way of intramolecular exchange, predominates over integrationof DNA into the genome, by way of intermolecular site-specificrecombination.

U.S. Pat. No. 5,928,914 (Leboulch, et al.) describes methods andcompositions for transforming cells, resulting in efficient and stablesite-specific integration of transgenes. Transformation is achieved byintroducing into a cell an acceptor vector, preferably a retroviralvector, which integrates into the genome of the cell. The acceptorvector comprises two incompatible lox sequences, L1 and L2. A donorvector is then introduced into the cell comprising a transgene flankedby the same L1 and L2 sequences. Stable gene transfer is initiated bycontacting the lox L1 and L2 sequences with Cre recombinase.

SUMMARY OF THE INVENTION

In one aspect, the invention provides an expression vector comprising(a) a first polynucleotide encoding a first, crippled, selectable marker(b) a second polynucleotide encoding a heterologous polypeptide ofinterest; and (c) a third polynucleotide encoding a second, amplifiableselectable marker. Suitable first selectable markers include sequencescoding antibiotic (e.g., neomycin) resistance containing one or morecrippling mutations. In one embodiment, the amplifiable selectablemarker is dihydrofolate reducatase (dhfr).

The invention also includes the following constructs: a plasmiddesignated pESN1dhfr; a plasmid designated pESN2dhfr; plasmid designatedpESN3dhfr; a plasmid designated pneo*dhfr5′del (e.g., pneo1dhfr5′del,pneo2dhfr5′del, pneo3dhfr5′del); and a plasmid designated pdhfr3′del.

In another aspect the invention includes a method for producing apolypeptide of interest in a host cell, comprising

(a) introducing an expression vector or construct described herein intoa host cell,

(b) selecting host cells which express the first and second selectablemarkers under conditions that select for stably integrated expressionvectors,

(c) growing the stably-transfected host cells under conditions whichfavor expression of the polypeptide of interest, and

(d) isolating the polypeptide of interest.

In certain embodiments, the heterologous polypeptide is a viral protein(e.g., an HIV protein) or is CAB2 or CAB4 and the host cell is amammalian or insect cell. Host cell lines that produces a polypeptide ofinterest using this method are also included in the present invention.

In another aspect, the invention includes a transfection systemcomprising

(a) a first construct comprising, in a suitable backbone, a sequenceencoding a first selectable marker and a sequence encoding a secondselectable marker, wherein the second selectable marker contains atleast one disabling mutation in its coding sequence; and

(b) a second construct comprising, in a suitable backbone, apolynucleotide sequence of interest and a sequence encoding a thirdselectable marker, wherein the third selectable marker is the sameselectable marker as the second selectable marker except that the thirdselectable marker contains at least one disabling mutation that is in adifferent region of the coding sequence than the disabling mutation insaid second selectable marker. In certain embodiments, the firstselectable marker encodes for antibiotic resistance, for example, byencoding wild-type or functionally impaired neomycin phosphotransferaseII, the second selectable marker encodes dhfr which disabled by at leastone mutation in the 5′ coding region and the third selectable markerencodes dhfr which is disabled by at least one different mutation in the3′ coding region. The disabling mutations may be, by way of example,point mutations or deletions. Where the constructs are plasmids, thetransfection system may further comprise

(c) first, second, and third promoters operably linked to said first andsecond selectable markers and said transgene, respectively;

(d) sequences encoding polyadenylation sites operably linked to saidfirst and second selectable markers and said transgene; and

(e) sequence encoding origins of replication operably linked to saidfirst, second selectable markers and said transgene. Promoters such asCMV promoter, an RSV promoter or an SV-40 early promoter may be used andeach sequence may be operably linked to a different promoter.

In another aspect, the invention includes a method for producing amammalian cell line for expression of a selected polynucleotidesequence, comprising

(a) introducing into a selected mammalian cell, having a genome, a firstconstruct comprising a sequence encoding a first selectable marker and asequence encoding a second selectable marker, wherein the secondselectable marker contains at least one disabling mutation in its codingsequence,

(b) selecting for a mammalian cell expressing the first selectablemarker, wherein said first construct stably integrates into the genome;

(c) introducing into the mammalian cell a second construct comprising apolynucleotide sequence of interest and a sequence encoding a thirdselectable marker, wherein the third selectable marker is the sameselectable marker as the second selectable marker except that the thirdselectable marker contains at least one disabling mutation that is in adifferent region of the coding sequence than the disabling mutation insaid second selectable marker; and

(d) selecting for a mammalian cell expressing a functional productencoded by the second selectable marker, wherein the functional productis encoded by a sequence produced by a recombination event between saidsecond and third selectable markers, and the resulting mammalian cell iscapable of expressing the selected polynucleotide sequence. In certainembodiments, the selected polynucleotide sequence encodes a polypeptideand expressing the selected polynucleotide sequence results inexpression of the polypeptide. Mammalian cells produced by this methodare also provided.

In another aspect, the invention includes a method for producing apolypeptide of interest in a host mammalian cell, said methodcomprising:

(a) introducing into said cell, having a genome, a first constructcomprising a sequence encoding a first selectable marker and a sequenceencoding a second selectable marker, wherein the selectable markercontains at least one disabling mutation in its coding sequence;

(b) selecting for a mammalian cell expressing the first selectablemarker, wherein said first construct stably integrates into the genome;

(c) introducing into the mammalian cell a second construct comprising apolynucleotide sequence encoding the polypeptide of interest and asequence encoding a third selectable marker, wherein the thirdselectable marker is the same selectable marker as the second selectablemarker except that the third selectable marker contains at least onedisabling mutation that is in a different region of the coding sequencethan the disabling mutation in said second selectable marker;

(d) selecting for a mammalian cell expressing a functional productencoded by the second selectable marker, wherein the functional productis encoded by a sequence produced by a recombination event between saidsecond and third selectable markers, and the resulting mammalian cell iscapable of expressing the polypeptide of interest; and

culturing the mammalian cell under conditions to produce the polypeptideof interest.

In one embodiment, the first selectable marker encodes for neomycinresistance and second and third selectable markers encode dhfr. Incertain embodiments, the constructs can be introduced into said cells byelectroporation or by calcium phosphate transfection. A mammalian cellline that produced according to these methods are also included.

These and other embodiments will be readily apparent to one skilled inthe art in light of the teachings of this specification.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention will be more fully described with reference to thedrawings in which:

FIG. 1 depicts the nucleotide sequence of CAB2 (SEQ ID NO:31). Lowercase represents original sequence.

FIG. 2 is a schematic representation of expression vectors designatedpESNdhfr, pESN1dhfr, pESN2dhfr and pESN3dhfr. The vectors differ in thelocation and/or number of mutations in the Neo gene. pESNdhfr contains awild-type Neo sequence. pESN1dhfr contains a mutation which alters aminoacid residue 182 of the Neo gene product. pESN2dhfr contains a mutationwhich alters amino acid residue 261 of the Neo gene product andpESN3dhfr contains mutations which alter both amino acid residues 182and 261 of the Neo gene product.

FIG. 3 is a schematic depicting an expression vector designated pcDNA3.The vector is 5446 nucleotides and contains, in a pUC19 vector backbone(bases 4450-5310), the following sequences: a CMV promoter (bases209-863); a T7 promoter (bases 864-882); a polylinker (bases 889-994); aSp6 promoter (bases 999-1016); a BGH polyA sequence (bases 1018-1249);and SV40 promoter (bases 1790-2215); an SV40 origin of replication(bases 1984-2069); an open reading frame (ORF) encoding neomycinresistance (Ne^(r)) (bases 2151-2932); an SV40 polyA sequence (bases3120-3250); and an ORF encoding ampicillin resistance (Amp^(r)) (bases4450-5310).

FIG. 4 is a schematic representation of a DNA construct designatedpneo*dhfr*1, which comprises a mutant neomycin resistance gene (neo*)and a disabled DHFR marker gene, where XX represent mutations in theDHFR gene.

FIG. 5 depicts a vector designated pXdhfr*2, which also carries atransgene and a disabled DHFR gene mutated at a different site asindicated by XX.

FIG. 6 depicts homologous recombination between pXdhfr*2 and pneo*dhfr1at the two copies of the disabled DHFR marker gene.

FIG. 7 shows the product of homologous recombination in which thedisabled DHFR gene has been rescued and the transgene is positioned inthe high expression locus of a mammalian cell genome by insertion of thetransgene proximate to the mutant neo* gene.

FIG. 8 depicts the pdhfr*2 DNA construct; a transgene, such as arecombinant protein gene, can be inserted into the polylinker of pdhfr*2to create pXdhfr*2.

FIG. 9 depicts a selection scheme, wherein the DNA construct identifiedas pdhfr3′del employs a deletion mutant of a DHFR gene and insertion ofa transgene in a polylinker forms the construct designated pXdhfr3′del(e.g., pdhfr3′del).

FIG. 10 shows a DNA construct pneo*dhfr5′del (e.g., pneo1dhfr5′del,pneo2dhfr5′del, pneo3dhfr5′del) in which a DHFR gene is disabled by 5′deletions and the disabled gene is covalently linked to a mutant neo*gene.

DETAILED DESCRIPTION

The invention provides novel expression vectors that allow stable,high-level expression of a polypeptide of interest in a host cell,particularly mammalian cells. The constructs comprise (i) a firstsequence encoding a first selectable marker; (ii) a second sequenceencoding a polypeptide product of interest (e.g., “transgene”); (iii) athird sequence encoding a second selectable marker. Typically, the threesequences are operably linked to at least one strong promoter, forexample a CMV promoter to increase expression and promote correctsplicing of the product of interest.

The invention also includes a transfection system for mammalian cellsusing the constructs described herein. The system uses first and secondconstructs as described above, although each construct has a disablingmutation in the second selectable marker. The disabling mutations are indifferent regions of the second selectable marker so that homologousrecombination between the two produces a functional product. This systemallows for high expression of a desired polypeptide. Mammalian cellsmodified by the transfection system described herein are also provided,as are methods for making these transfected cells.

Thus, the invention described herein eliminates the tedious process ofidentification of high expression loci in mammalian cells and providesan efficient mechanism by which any desired polypeptide can be expressedat high levels using the novel cell lines generated as described herein.

Definitions

A “nucleic acid” molecule can include, but is not limited to,procaryotic sequences, eucaryotic mRNA, CDNA from eucaryotic mRNA,genomic DNA sequences from eucaryotic (e.g., mammalian) DNA, and evensynthetic DNA sequences. The term also captures sequences that includeany of the known base analogs of DNA and RNA. A “coding sequence” or asequence which “encodes” a selected polypeptide, is a nucleic acidmolecule which is transcribed (in the case of DNA) and translated (inthe case of mRNA) into a polypeptide in vivo when placed under thecontrol of appropriate regulatory sequences (or “control elements”). Theboundaries of the coding sequence are determined by a start codon at the5′ (amino) terminus and a translation stop codon at the 3′ (carboxy)terminus. A coding sequence can include, but is not limited to, cDNAfrom viral, procaryotic or eucaryotic mRNA, genomic DNA sequences fromviral or procaryotic DNA, and even synthetic DNA sequences. Atranscription termination sequence may be located 3′ to the codingsequence.

Typical “control elements”, include, but are not limited to,transcription promoters, transcription enhancer elements, transcriptiontermination signals, polyadenylation sequences (located 3′ to thetranslation stop codon), sequences for optimization of initiation oftranslation (located 5′ to the coding sequence), and translationtermination sequences. “Operably linked” refers to an arrangement ofelements wherein the components so described are configured so as toperform their usual function. Thus, a given promoter operably linked toa coding sequence is capable of effecting the expression of the codingsequence when the proper enzymes are present. The promoter need not becontiguous with the coding sequence, so long as it functions to directthe expression thereof. Thus, for example, intervening untranslated yettranscribed sequences can be present between the promoter sequence andthe coding sequence and the promoter sequence can still be considered“operably linked” to the coding sequence.

“Recombinant” as used herein to describe a nucleic acid molecule means apolynucleotide of genomie, CDNA, semisynthetic, or synthetic originwhich, by virtue of its origin or manipulation: (1) is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature; and/or (2) is linked to a polynucleotide other than that towhich it is linked in nature. The term “recombinant” as used withrespect to a protein or polypeptide means a polypeptide produced byexpression of a recombinant polynucleotide. “Recombinant host cells,”“host cells,” “cells,” “cell lines,” “cell cultures,” and other suchterms denoting procaryotic microorganisms or eucaryotic cell linescultured as unicellular entities, are used interchangeably, and refer tocells which can be, or have been, used as recipients for recombinantvectors or other transfer DNA, and include the progeny of the originalcell which has been transfected. It is understood that the progeny of asingle parental cell may not necessarily be completely identical inmorphology or in genomic or total DNA complement to the original parent,due to accidental or deliberate mutation. Progeny of the parental cellwhich are sufficiently similar to the parent to be characterized by therelevant property, such as the presence of a nucleotide sequenceencoding a desired peptide, are included in the progeny intended by thisdefinition, and are covered by the above terms.

Techniques for determining nucleic acid and amino acid “sequenceidentity” also are known in the art. Typically, such techniques includedetermining the nucleotide sequence of the mRNA for a gene and/ordetermining the amino acid sequence encoded thereby, and comparing thesesequences to a second nucleotide or amino acid sequence. In general,“identity” refers to an exact nucleotide-to-nucleotide or aminoacid-to-amino acid correspondence of two polynucleotides or polypeptidesequences, respectively. Two or more sequences (polynucleotide or aminoacid) can be compared by determining their “percent identity.” Thepercent identity of two sequences, whether nucleic acid or amino acidsequences, is the number of exact matches between two aligned sequencesdivided by the length of the shorter sequences and multiplied by 100. Anapproximate alignment for nucleic acid sequences is provided by thelocal homology algorithm of Smith and Waterman, Advances in AppliedMathematics 2:482-489 (1981). This algorithm can be applied to aminoacid sequences by using the scoring matrix developed by Dayhoff, Atlasof Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl.3:353-358, National Biomedical Research Foundation, Washington, D.C.,USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763(1986). An exemplary implementation of this algorithm to determinepercent identity of a sequence is provided by the Genetics ComputerGroup (Madison, Wis.) in the “BestFit” utility application. The defaultparameters for this method are described in the Wisconsin SequenceAnalysis Package Program Manual, Version 8 (1995) (available fromGenetics Computer Group, Madison, Wis.). A preferred method ofestablishing percent identity in the context of the present invention isto use the MPSRCH package of programs copyrighted by the University ofEdinburgh, developed by John F. Collins and Shane S. Sturrok, anddistributed by IntelliGenetics, Inc. (Mountain View, Calif.). From thissuite of packages the Smith-Waterman algorithm can be employed wheredefault parameters are used for the scoring table (for example, gap openpenalty of 12, gap extension penalty of one, and a gap of six). From thedata generated the “Match” value reflects “sequence identity.” Othersuitable programs for calculating the percent identity or similaritybetween sequences are generally known in the art.

Two nucleic acid fragments are considered to “selectively hybridize” asdescribed herein. The degree of sequence identity between two nucleicacid molecules affects the efficiency and strength of hybridizationevents between such molecules. A partially identical nucleic acidsequence will at least partially inhibit a completely identical sequencefrom hybridizing to a target molecule. Inhibition of hybridization ofthe completely identical sequence can be assessed using hybridizationassays that are well known in the art (e.g., Southern blot, Northernblot, solution hybridization, or the like, see Sambrook, et al., supraor Ausubel et al., supra). Such assays can be conducted using varyingdegrees of selectivity, for example, using conditions varying from lowto high stringency. If conditions of low stringency are employed, theabsence of non-specific binding can be assessed using a secondary probethat lacks even a partial degree of sequence identity (for example, aprobe having less than about 30% sequence identity with the targetmolecule), such that, in the absence of non-specific binding events, thesecondary probe will not hybridize to the target.

When utilizing a hybridization-based detection system, a nucleic acidprobe is chosen that is complementary to a target nucleic acid sequence,and then by selection of appropriate conditions the probe and the targetsequence “selectively hybridize,” or bind, to each other to form ahybrid molecule. A nucleic acid molecule that is capable of hybridizingselectively to a target sequence under “moderately stringent” typicallyhybridizes under conditions that allow detection of a target nucleicacid sequence of at least about 10-14 nucleotides in length having atleast approximately 70% sequence identity with the sequence of theselected nucleic acid probe. Stringent hybridization conditionstypically allow detection of target nucleic acid sequences of at leastabout 10-14 nucleotides in length having a sequence identity of greaterthan about 90-95% with the sequence of the selected nucleic acid probe.Hybridization conditions useful for probe/target hybridization where theprobe and target have a specific degree of sequence identity, can bedetermined as is known in the art (see, for example, Nucleic AcidHybridization: A Practical Approach, editors B. D. Hames and S. J.Higgins, (1985) Oxford; Washington, D.C.; IRL Press).

With respect to stringency conditions for hybridization, it is wellknown in the art that numerous equivalent conditions can be employed toestablish a particular stringency by varying, for example, the followingfactors: the length and nature of probe and target sequences, basecomposition of the various sequences, concentrations of salts and otherhybridization solution components, the presence or absence of blockingagents in the hybridization solutions (e.g., formamide, dextran sulfate,and polyethylene glycol), hybridization reaction temperature and timeparameters, as well as, varying wash conditions. The selection of aparticular set of hybridization conditions is selected followingstandard methods in the art (see, for example, see Sambrook, et al.,supra or Ausubel et al., supra)

A first polynucleotide is “derived from” second polynucleotide if it hasthe same or substantially the same basepair sequence as a region of thesecond polynucleotide, its cDNA, complements thereof, or if it displayssequence identity as described above. A first polypeptide is “derivedfrom” a second polypeptide if it is (i) encoded by a firstpolynucleotide derived from a second polynucleotide, or (ii) displayssequence identity to the second polypeptides as described above.

“Encoded by” refers to a nucleic acid sequence which codes for apolypeptide sequence, wherein the polypeptide sequence or a portionthereof contains an amino acid sequence of at least 3 to 5 amino acids,more preferably at least 8 to 10 amino acids, and even more preferablyat least 15 to 20 amino acids from a polypeptide encoded by the nucleicacid sequence. Also encompassed are polypeptide sequences which areimmunologically identifiable with a polypeptide encoded by the sequence.

“Purified polynucleotide” refers to a polynucleotide of interest orfragment thereof which is essentially free, e.g., contains less thanabout 50%, preferably less than about 70%, and more preferably less thanabout 90%, of the protein with which the polynucleotide is naturallyassociated. Techniques for purifying polynucleotides of interest arewell-known in the art and include, for example, disruption of the cellcontaining the polynucleotide with a chaotropic agent and separation ofthe polynucleotide(s) and proteins by ion-exchange chromatography,affinity chromatography and sedimentation according to density.

The term “transfection” is used to refer to the uptake of foreign DNA bya cell. A cell has been “transfected” when exogenous DNA has beenintroduced inside the cell membrane. A number of transfection techniquesare generally known in the art. See, e.g., Graham et al. (1973)Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratorymanual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986)Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene13:197. Such techniques can be used to introduce one or more exogenousDNA moieties into suitable host cells. The term refers to both stableand transient uptake of the genetic material, and includes uptake ofpeptide- or antibody-linked DNAs.

“Nucleic acid expression vector” or “Expression cassette” refers to anassembly which is capable of directing the expression of a sequence orgene of interest. The nucleic acid expression vector includes a promoterwhich is operably linked to the sequences or gene(s) of interest. Othercontrol elements may be present as well. Expression cassettes describedherein may be contained within a plasmid construct. In addition to thecomponents of the expression cassette, the plasmid construct may alsoinclude a bacterial origin of replication, one or more selectablemarkers, a signal which allows the plasmid construct to exist assingle-stranded DNA (e.g. a M13 origin of replication), a multiplecloning site, and a “mammalian” origin of replication (e.g., a SV40 oradenovirus origin of replication). Vector backbones are discussed infurther detail below. A “vector” is capable of transferring genesequences to target cells (e.g., viral vectors, non-viral vectors,particulate carriers, and liposomes). Typically, “vector construct,”“expression vector,” and “gene transfer vector,” mean any nucleic acidconstruct capable of directing the expression of a gene of interest andwhich can transfer gene sequences to target cells. Thus, the termincludes cloning and expression vehicles, as well as viral vectors.

A “selectable marker” or “reporter marker” refers to a nucleotidesequence included in a gene transfer vector that has no therapeuticactivity, but rather is included to allow for simpler preparation,manufacturing, characterization or testing of the gene transfer vector.Suitable selectable markers are discussed further below.

Expression Vectors

In one embodiment, the expression vectors comprise polynucleotidesencoding (i) a first crippled, selectable marker; (ii) a product ofinterest, also referred to as the “transgene”; and (iii) a second,amplifiable selectable marker. By “crippled” is meant a sequenceencoding a selectable marker that includes one or more mutations thatdiminish, but do not destroy, the function of the marker. In oneembodiment the first crippled selectable marker is a neomycin resistance(Neo^(r)) sequence in which amino acid residue 182 (Glu) is mutated toAsp. (see, FIG. 2, pESN1.dhfr and Yanofsky et al., infra). In anotherembodiment, the first crippled selectable marker is a Neo^(r) sequencein which amino acid residue 261 (Asp) is mutated to Asn (N). (FIG. 2,pESN2.dhfr). In yet another embodiment, the first crippled selectablemarker is a Neo sequence containing both the above described mutations,e.g., Glu182 to Asp and Asp261 to Sn. (See, FIG. 2, pESN3,dhfr). Othersuitable selectable markers and methods of making and testing cripplingmutations are described below.

Also included in the expression vectors is a sequence encoding a productof interest (i.e., a “transgene”). Suitable transgenes includepolynucleotides encoding for any polypeptide of interest. In oneembodiment, the transgene encodes a chimeric protein such as “CAB”. CABcombines features of membrane co-factor protein (MTP) and decayaccelerating factor (SAF) to inhibit complement activation. See, e.g.,Higgins et al. (1997) J. Immunology 158(6):2872-2881. In certainembodiments, the sequence encoding the transgene can also be designed,as described herein, such that aberrant mRNA splicing of the product isreduced or eliminated.

The expression vectors also include a second selectable, amplifiablemarker. The second amplifiable marker facilitates selection of thosecells which express the transgene at high levels, for exampledihydrofolate reducatase (DHFR). Suitable markers are discussed indetail below.

The expression vectors of the present invention can be producedfollowing the teachings of the present specification in view oftechniques known in the art. Sequences, for example encoding transgenes,may be commercially available, for example, green fluorescent protein(G.P.) is available from Clontech, Palo Alto, Calif., and lucifers isavailable from Promega, Madison, Wis. Exemplified herein are expressionvectors which direct high-level expression of the chimeric protein CABand expression of HIV proteins. However, it is to be understood that theexpression vectors, constructs and methods of the present invention areapplicable to high-level expression of any polypeptide of interest.

Another standard source for the polynucleotides used in the inventionis, of course, polynucleotides isolated from an organism (e.g,bacteria), a cell, or selected tissue. Nucleic acids from the selectedsource can be isolated by standard procedures, which typically includesuccessive phenol and phenol/chloroform extractions followed by ethanolprecipitation. After precipitation, the polynucleotides can be treatedwith a restriction endonuclease which cleaves the nucleic acid moleculesinto fragments. Fragments of the selected size can be separated by anumber of techniques, including agarose or polyacrylamide gelelectrophoresis or pulse field gel electrophoresis (Care et al. (1984)Nuc. Acid Res. 12:5647-5664; Chu et al. (1986) Science 234:1582; Smithet al. (1987) Methods in Enzymology 151:461), to provide an appropriatesize starting material for cloning.

Another method of obtaining the nucleotide components of the expressionvectors or constructs is PCR. General procedures for PCR are taught inMacPherson et al., PCR: A PRACTICAL APPROACH,(IRL Press at OxfordUniversity Press, (1991)). PCR conditions for each application reactionmay be empirically determined. A number of parameters influence thesuccess of a reaction. Among these parameters are annealing temperatureand time, extension time, Mg²⁺ and ATP concentration, pH, and therelative concentration of primers, templates and deoxyribonucleotides.Exemplary primers are described below in the Examples. Afteramplification, the resulting fragments can be detected by agarose gelelectrophoresis followed by visualization with ethidium bromide stainingand ultraviolet illumination.

Another method for obtaining polynucleotides is by enzymatic digestion.For example, nucleotide sequences can be generated by digestion ofappropriate vectors with suitable recognition restriction enzymes.Restriction cleaved fragments may be blunt ended by treating with thelarge fragment of E. coli DNA polymerase I (Klenow) in the presence ofthe four deoxynucleotide triphosphates (dNTPs) using standardtechniques.

Polynucleotides are inserted into suitable backbones, for example,plasmids, using methods well known in the art. For example, insert andvector DNA can be contacted, under suitable conditions, with arestriction enzyme to create complementary or blunt ends on eachmolecule that can pair with each other and be joined with a ligase.Alternatively, synthetic nucleic acid linkers can be ligated to thetermini of a polynucleotide. These synthetic linkers can contain nucleicacid sequences that correspond to a particular restriction site in thevector DNA. Other means are known and available in the art. A variety ofsources can be used for the component polynucleotides.

Constructs for Transfection Systems

The invention also includes using first and second constructs inparticular methods of producing and selecting stably transfected celllines, particularly mammalian cell lines. In the transfection systemdescribed herein, the first construct comprises polynucleotides encodingfirst and second selectable markers. The first selectable marker istypically under the control of a strong promoter. As described above,the first selectable marker preferably contains at least one cripplingmutation. In addition, in these embodiments, the second marker containsa disabling mutation, preferably in the 3′ or 5′ end of the codingregion. By “disabling” is meant a mutation that reduces or, preferablyeliminates, a functional gene product.

An exemplary first construct is shown in FIG. 4 and identified aspneo*dhfr*1. The first selectable marker encoded by this construct is anantibiotic resistance marker which imparts resistance to neomycin(neo*), preferably a crippled mutant of neo, designated neo* in theFigures. The neo* gene is operably linked to a strong promoter (e.g.,CMV or RSV, shown 3′ to the neo* gene), and may be generally linked toother expression regulatory elements such as polyadenylation sequences(e.g., a bovine growth hormone (BGH) polyadenylation site). Further, anorigin of replication useful for propagation in a microorganism may alsobe included (e.g., the f1 ori). The BGH polyA sequence and the f1 originare typically 5′ to the neo* gene.

In this example, the second selectable marker of pneo*dhfr*1 constructencodes, for example, dihydrofolate reductase (DHFR). However, thefunction of the second selectable marker is disabled. Referring to FIG.4, XX represent disabling mutations proximate the 5′ end of the DHFRgene. The DHFR gene is driven by the SV40 early promoter and, at its 3′end, are sequences coding for SV40 polyA, ColEl, AmpR, and anappropriate restriction site, such as BglIII shown in FIG. 4.

The second vector construct of the transfection system comprises apolynucleotide encoding a heterologous polypeptide of interest (labeled“transgene” in the Figures) and a polynucleotide encoding the samesecond marker (i.e., the “second” selectable marker)that is used in thefirst construct. However, in the second construct, the second markersequence contains a disabling mutation in a different position relativeto the first construct, preferably on the opposite end of the codingregion. The location of the disabling mutation in the second selectablemarker of the first construct, and the location at the disablingmutation in the selectable marker of the second construct, are such thata single recombination event between the two copies of the codingsequences of the selectable marker can generate a functional selectablemarker. Once the first construct has randomly integrated into the genomeof the host cell homologous recombination events between the integratedfirst construct and second construct results in insertion of thetransgene under the control of the strong promoter. Functionalrecombinants can be readily selected by assaying for the second marker.Thus, unlike other targeting systems, for example, as described inDetloff et al. (1994) Molecular and Cellular Biology 14:3936-6941, thetransfection system described herein does not rely on homologousrecombination events to alter the transgene of interest.

An exemplary second construct is shown in FIG. 5, and is designatedpXdhfr*2. Here, the transgene is operably linked to the CMV promoter andincludes a 3′ BGH polyA tail and f1 origin of replication. The secondselectable marker, DHFR, is disabled by mutation(s) at the opposite endof the coding region to the disabled marker in the first construct.Thus, in FIG. 5, the mutations (designated XX) are in the 3′ end of theDHFR sequence. As in the pneo*dhfr*1 construct, the disabled DHFR markeris followed by nucleotide sequences encoding SV40 polyA, ColEl , AmpR,and a matching restriction site, in this case BglIII.

First and second constructs for use in transfection systems can beproduced following the guidance of the present specification usingmethods known in the art, for example as described above.

General Methods

The expression vectors and methods described herein are useful inimproving expression of a transgene of interest. Stable, high-levelexpression of genes in mammalian cells is critically dependent on boththe site of integration and the copy number. The selection pressureimposed by the usual concentration of the neomycin analog G418, forexample, is low and yield cell clones with widely different expressionlevels. Aberrant splice sites with the transgene may also hamperexpression. The expression vectors described herein solve this and otherproblems by using a crippled first selectable marker operably linked atransgene of interest and a second, amplifiable selectable marker,which, in certain embodiments, contains a disabling mutation. Thecrippled marker and second marker allow for selection of high-expressingclones. In addition, expression may be increased by altering thetransgene such that aberrant slicing is corrected. A strong promoteroptionally containing an intron and/or enhancer may also be added topromote expression of the transgene.

The invention also provides a novel transfection system using first andsecond constructs. These highly efficient methods are capable ofgenerating cells which produce high levels of the desired polypeptide.Cells are transformed with the first construct which integrates randomlyinto the target cellular genome. Exemplary target cells include, but arenot limited to, mammalian cells and call lines (e.g., BHK, VERO, HT1080,293, RD, COS-7, and CHO cells). The cells are grown in the presence ofthe appropriate substrate for the first selectable marker, for example,G418 if the first selectable marker encodes neomycin. Cells that surviveselection in high concentration of the antibiotic have integrated theneomycin resistance gene at a high expression locus.

The cells having an integrated first construct are then used to make thecell lines of the invention by the following technique. The secondconstruct is introduced into cells carrying an integrated copy of thefirst-construct. Homologous recombination between the two disabled DHFRgenes results in a functional DHFR gene, as well as insertion of thedesired gene encoding the functional polypeptide at the high expressionlocus (see, FIG. 7). Cell lines that survive selection with theappropriate toxin or antibiotic (e.g., methotrexate) have successfullyundergone homologous recombination.

The second step can be repeated with any number of variations in thedesired gene encoding the functional polypeptide (i.e. transgene) inorder to obtain high level expression of the polypeptide of interest.This technique will now be described in greater detail with reference tothe Figures.

Transgenes

The transfection system described herein is useful to express anypolypeptide of interest. As used herein, the term “transgene” refers toexogenous DNA inserted in the genome of a mammalian cell whoseexpression at an elevated level in the cell is desired. The transgeneemployed in this invention encodes a functional polypeptide, which is anamino acid sequence that possesses a biological activity or an aminoacid sequence that is a precursor of a protein having a biologicalactivity. The transgene will generally encode a native or recombinantprotein, although the expression of other polypeptides, such as epitopesor other immunologically active polypeptides, arc contemplated withinthe scope of this invention. Examples of proteins that can be expressedusing the method of this invention are hormones; cytokines, such asgrowth factors; enzymes; receptors; oncogenes; polypeptide vaccines,viral proteins, and structural and secretory proteins. In oneembodiment, the expression vectors contain sequences encoding CAB2,described above. These sequences have been further modified such thataberrant splicing is corrected, see Examples. In another embodiments,the expression vectors contain sequences encoding viral polypeptides,particular derived from HIV. These viral polypeptides include, but arenot limited to, envelope (Env) polypeptides (e.g., gp120, gp160, gp140,gp41, and monomers or multimers thereof), Gag polypeptides (e.g., Gag,Gag-protease, Gag-polymerase), rev, tat, etc. In addition, theexpression vectors can include transgenes encoding for synthetic HIVpolypeptides, for example constructs described in the Examples.

The transgene employed in the constructs of the invention can be clonedsequences that retain intronic regions. If the exonic structure of thegene is known, the coding exons can be inserted in the constructs.

Expression of the polypeptide of interest can be directed by a promoterhomologous to the polypeptide coding sequences (for example, humanglucose-6-phosphate dehydrogenase under the control of its owntranscription promoter sequences). Further, other homologous orheterologous expression control elements (e.g., affecting transcription,translation, or post-translational events) may be used.

It should be understood that expression of the transgene in themammalian cells of the invention can be stable or transient. Eventransient expression at a higher than normal level is useful forfunctional studies in the cells or for the production and recovery ofproteins of interest.

Selectable Markers

Any suitable sequence encoding for a selectable marker can be used asthe first or second markers in the compositions and methods describedherein. Typically, the selectable marker genes employed in thisinvention can be obtained from readily available sources.

In one embodiment of the invention (depicted in the accompanyingFigures), the first selectable marker encodes a gene which confersresistance to antibiotics. For example, in one aspect, the firstselectable marker comprises a neomycin (neo) resistance gene. Inpreferred embodiments, mutant neo genes, designated neo*, can be used toestablish a high expression locus in chromosomal DNA of the mammaliancalls. (see, e.g., Yanofsky et al. 9(1990) PNAS USA 87:3435-39). Theneomycin resistance gene of transposon Tn5 encodes for neomycinphosphotransferase II, which confers resistance to various antibiotics,including G418 and kanamycin. A single base substitution significantlyimpairs the function of the enzyme. Neo resistance genes used asselectable markers can be identified by restriction enzyme digestion,because base change results in the loss of an XhoII restriction site.See also Blazquez et al., (Molecular Microbiology, (2991)5(6):1511-1518) for techniques for creating mutant neo genes for use inthis invention. The optimum amount of substrate (e.g., G418) needed forselection can be individually determined for each cell line. Othersimilar selectable markers include, but are not limited to, those listedbelow.

Temperature-sensitive selectable markers can also be employed in thepractice of the invention. For example, temperature-sensitive neo willbe nearly wild type in function at non-stringent temperature and havelow activity at stringent temperature. After electroporation and initialselection of pneo*ts-dhfr*1, insertion can be performed using G418 atnon-stringent temperature, After colonies begin to grow, stringenttemperature can be used to kill off colonies carrying low expressioninsertions.

As described above, the second selectable marker is included to enhanceexpression. In the expression vectors, the second selectable marker istypically an amplifiable marker, for example, DHFR. DHFR is necessaryfor purine biosynthesis. In the absence of erogenous purines, thisenzyme is required for growth. Methotrexate is a potent competitiveinhibitor of DHFR, so increasing methotrexate concentration selects forcells that express increased levels of DHFR. Extremely high levels ofexpression of the transfected normal DHFR gene are needed for selectionin cell lines with high endogenous DHFR levels. To increase expressionto even, higher levels, the transgene can be amplified by standard DHFRamplification methods.

For the constructs used in the transfection methods, the secondselectable marker is found on both the first and second constructs ofthe claimed transfection system. However, these copies of the secondselectable marker are disabled by mutations in different regions. Thesemutations are such that homologous recombination between the two mutatedcopies will rescue function of the gene. In one embodiment, the second(disabled) selectable marker encodes for DHFR.

The disabling mutations to the second selectable marker of the first andsecond constructs can be introduced by a variety of methods, forexample, the DHFR gene or other marker gene can be disabled by thedeletion of nucleotides in the native gene sequence or by substitutionof nucleotides. Bullerjahn et al. (J. of Bio Chem. (1992) 267:864-870)provide an overview of how the activity of dihydrofolate reductase (DMM)enzyme is affected by residue deletions and/or substitutions. Theydescribe deletions in critical areas (achieved by expressing an alteredgene), which result in reduction of activity with complete loss ofactivity following deletion of 6 amino acids. In addition, a mutant DHFRgene is available that encodes an enzyme resistant to methotrexate(Simonsen et al., (1983) PNAS USA 80:2495-2499).

In some instances, it may also be desirable to amplify the gene encodingthe second selectable marker (e.g., DHFR). This can be accomplished, forexample, by increasing the concentration of substrate (e.g.,methotrexate). Thus, cells harboring copies of the DHFR gene, multipliedmany times, can be selected by sequential increases in the concentrationof methotrexate to high levels. This technique also makes it possible toidentify stable transformants. That is, whereas this phenotype isfrequently unstable and is lost after several cell cycles in the absenceof selective pressure, the unstable configuration of DHFR genes becomesstable under continuous selective pressure. Stably amplified cellscontain the amplified DHFR genes within their chromosomes.

It will be understood that other selectable markers, which permitisolation of stable transfectants, can be employed in this invention aseither first or second (disabled) markers. An example of anotherselectable marker is adenosine deaminase (ADA). A medium supplementedwith thymidine, 9-β-D-xylofuranosyl adenine (Xyl-A), and2′-deoxycoformycin (dCF) is employed. Xyl-A can be converted to Xyl-ATPand incorporated into nucleic acids, resulting in cell death. Xyl-A isdetoxified to its inosine derivative by ADA. dCF is a transition stateanalogue inhibitor of ADA, and is needed to inactivate ADA endogenous tothe parental cell type. As the level of endogenous ADA varies with celltype, the appropriate concentration of dCP for selection will vary aswell. Kaufman et al., (1986) PNAS USA, 83:3136-3140. ADA-deficient CHOcells are also available.

Another suitable selectable marker for use in the invention is thymidinekinase (TK). In forward selection (TK⁻ to TX⁺), complete medium issupplemented with hypoxanthine, aminopterin, thymidine, and glycine (HATmedium). In reverse selection (TK⁺ to TX⁻), complete medium issupplemented with 5-bromodeoxyuridine (BrdU). Under normal growthconditions, cells do not need thymidine kinase, because the usual meansfor synthesizing dTDP is through dCDP. Addition of BrdU to the mediumwill kill Tk⁺ cells, as BrdU is phosphorylated by TX and thenincorporated into DNA. Selection of TK⁺ cells in HAT medium is primarilydue to the presence of aminopterin, which blocks the formation of dTDPfrom dCDP. Cells, therefore, need to synthesize dTTP from thymidine, apathway that requires TK. Thymidine kinase is widely used in mammaliancell culture because both forward and reverse selection conditionsexist. Like ADA and DHFR, most mammalian cell lines express TK, removingthe possibility of using the marker in those lines unless BrdU is usedto select a TK⁻ mutant. See Littlefield et al., (1964)Science145:709-710.

An example of another suitable dominant selectable marker for use in theinvention is xanthine-guanine phosphoribosyltransferase (XGPRT, gpt).Medium containing dialyzed fetal calf serum and xanthine, hypoxanthine,thymidine, aminopterin, mycophenolic acid, and L-glutamine can beemployed. Aminopterin and mycophenolic acid both block the de novopathway for synthesis of GMP. Expression of XGPRT allows cells toproduce GMP from xanthine, allowing growth on medium that containsxanthine, but not guanine. XGPRT is a bacterial enzyme that does nothave a mammalian homolog, allowing XGPRT to function as a dominantselectable marker in mammalian cells. The amount of mycophenolic acidnecessary for selection varies with cell type and can be determined bytitration in the absence and presence of guanine. See Mulligan at al.,(1981) PNAS USA 78:2072-2076.

The selectable marker hygromycin-B-phosphotransferase (HPH) can also beemployed. Complete medium in supplemented with hygromycin-B.Hygromycin-B is an aminocyclitol that inhibits protein synthesis bydisrupting translocation and promoting mistranslation. The HPH gene hasbeen used in mammalian systems, and vectors that efficiently express thegene are available. See Gritz et al., (1983)Gene 25:179-188; and Palmeret al., (1987) PNAS USA 84:1055-1059.

Another useful marker is chloramphenicol resistance, Resistance ismediated by chloramphenicol acetyltransferase (CAT), which inactivateschloramphenicol by converting it into mono- and bi-acetylatedderivatives. These derivatives can be detected by thin layerchromatography. This enzyme is expressed in mammalian cells and iseasily detected because it does not naturally occur in mammalian cells.The gene can be obtained from a derivative of PBR322 carrying transposonTn9 by cleavage with suitable enzymes. It can be introduced into vectorpSV2, yielding plasmid pSV2-cat (ATCC Accession No. 37155) a plasmidexpresses the CAT gene from the early promoter of SV40.

Gossen et al., Science (1995) 268:1766-1769, describe fusion of atetracycline resistance gene repressor to a viral transcriptionactivation domain in order to induce rapid, greatly amplified geneexpression in the presence of tetracycline. It is a modification of apreexisting system in which low levels of tetracycline prevented geneexpression. The gene that codes for the tetracycline resistance generepressor was mutagenized and a mutant fusion protein was created thatdepended on tetracycline for activation was identified. The constructcan provide an on/off switch for high expression of a gene.

Another suitable marker is adeninephosphoribosyl transferase (APRT). Theenzyme APRT, another enzyme of the purine salvage pathway, catalyzes theconversion of adenine to AMP. APRT positive cells can be selectable in amedium containing, for example, the glutamine analogue azaserine, whichprevents de novo synthesis of purines. APRT-negative cells cannot begrown in a medium containing azaserine and adenine, and can be selectedby treatment with 2,6-diaminopurine. This compound is toxic for normalcells, but APRT-negative cells survive because they do not incorporateit.

The expression of the selectable marker coding sequences can be placedunder the control of, for example, promoter sequences derived from CMV,RSV, polyA′-BGH, SV40 or the like, and may include other expressioncontrol elements as well (e.g., sequences affecting transcription,translation or post-translation modifications).

Regulatory Sequences

In addition to selectable markers and transgenes, the constructsdescribed herein may contain suitable regulatory elements. Regulatoryelements (or control elements) are selected for use in the host cell ofinterest, for example, selectable markers may be included to allowpropagation in microorganisms, (e.g., f1 origin of replication andampicillin resistance encoding sequences). Such regulatory elementsinclude, but are not limited to, transcription promoters, transcriptionenhancer elements, transcription termination signals, polyadenylationsequences (located 3′ to the translation stop codon), sequences foroptimization of initiation of translation (located 5′ to the codingsequence), translation termination sequences, secretion signalsequences, and sequences that direct post-translational modification(e.g., glycosylation sites). Transcription promoters can includeinducible promoters (where expression of a polynucleotide sequenceoperably linked to the promoter is induced by an analyte, cofactor,regulatory protein, etc.), repressible promoters (where expression of apolynucleotide sequence operably linked to the promoter is induced by ananalyte, cofactor, regulatory protein, etc.), and constitutivepromoters.

The selectable markers each be sandwiched between the promoter and apolyadenylation site, such as BGH polyA or SV40 polyA as shown in theFigures. A chimeric mRNA is transcribed from the promoters andstabilized by the polyadenylation signals located 3′ to the codingregions. During construction of the mammalian transcription unit, anybacterial promoters in the plasmids can be replaced with transcriptionalregulatory sequences that are active in animal cells. Two such sequencesare shown in the Figures, the CMV promoter and SV40 early promoter.Non-limiting examples of other promoters that can be employed are RSVand HSV-TK.

Ordinarily, gene expression will be constitutive, although regulatablepromoters can be employed. Examples of suitable regulatable promotersare Tet, ecdysone and lac repressor sequences. Gene expression can alsobe controlled by transcription-regulation using heat, light, or metals,such as by the use of metallothionine genes or heat shock genes.

Exemplary “Backbone” Vectors

The above-described components of the transfection system of the presentinvention can be incorporated into a number of suitable backbone vectorsto facilitate manipulation of the expression vectors and constructs. Forexample, incorporation of the components into a vector containing meansthat allow replication in a microorganism greatly facilitatespropagation and isolation of the constructs (i.e., creating shuttlevectors). Exemplary backbone vectors include, but are not limited to,the following: pCMV6a and pUC19 (Example 1).

A variety of such backbone vectors are available for appropriate hostsystems. These systems include, but are not limited to, the following:baculovirus {Reilly, P. R., et al., BACULOVIRUS EXPRESSION VECTORS: ALABORATORY MANUAL (1992); Beames, et al., Biotechniques 11:378 (1991);Pharmingen; Clontech, Palo Alto, Calif.); pAcC13, a shuttle vector foruse in the Baculovirus expression system derived from pAcC12, MunemitsuS., et al., Mol Cell Biol. 10(11):5977-5982, 1990}, bacteria {pBR322;Ausubel, F. M., et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, JohnWiley and Sons, Inc., Media Pa.; Clontech; Promega, Madison, Wis.; LifeTechnologies, Gaithersburg, Md.}, yeast {Rosenberg, S. and Tekamp-Olson,P., U.S. Pat. No. RE35,749, issued, Mar. 17, 1998, herein incorporatedby reference; Shuster, J. R., U.S. Pat. No. 5,629,203, issued May 13,1997, herein incorporated by reference; Gellissen, G., et al., AntonieVan Leeuwenhoek, 62(1-2):79-93 (1992); Romanos, M. A., et al., Yeast8(6):423-488 (1992); Goeddel, D. V., Methods in Enzymology 185 (1990);Guthrie, C., and G. R. Fink, Methods in Enzymology 194 (1991)},mammalian cells {Clontech; Promega, Madison, Wis.; Life Technologies,Gaithersburg, Md.; e.g., Chinese hamster ovary (CHO) cell lines (Haynes,J., et al., Nuc. Acid. Res. 11:687-706 (1983); 1983, Lau, Y. F., et al.,Mol. Cell. Biol. 4:1469-1475 (1984); Methods in Enzymology, vol. 185,pp537-566. Academic Press, Inc., San Diego Calif. (1991)}, and plantcells {plant cloning vectors, Clontech Laboratories, Inc., Palo Alto,Calif., and Pharmacia LKB Biotechnology, Inc., Pistcataway, N. J.; Hood,E., et al., J. Bacteriol. 168:1291-1301 (1986); Nagel, R., et al., FEMSMicrobiol. Lett. 67:325 (1990); An, et al., “Binary Vectors”, and othersin Plant Molecular Biology Manual A3:1-19 (1988); Miki, B. L. A., etal., pp.249-265, and others in Plant DNA Infectious Agents (Hohn, T., etal., eds.) Springer-Verlag, Wien, Austria, (1987); Plant MolecularBiology: Essential Techniques, P. G. Jones and J. M. Sutton, New York,J. Wiley, 1997; Miglani, Gurbachan Dictionary of plant Genetics andMolecular Biology, New York, Food Products Press, 1998; Henry, R. J.,Practical Applications of Plant Molecular Biology, New York, Chapman &Hall, 1997 }.

Introduction of Expression Vectors and Constructs

The vectors and constructs described herein can be introduced intosuitable host cells by a variety of methods. In particular, themammalian cells that are modified by the process of this invention canbe obtained by transfection or infection with a vector. Transfection canbe carried out by well known techniques, such as calcium phosphatetransfection, DEAE-dextran mediated transfection, electroporation,liposome mediated transfection, or microinjection (Ausubel, et al.,supra). Exemplary methods are also described in the Examples.Transfection can be employed with DNA fragments that are unable toreplicate, or with DNA that is not readily packaged in viral vectors, orwhere infection of the mammalian cells with viral DNA is to be avoided.

Vectors can be derived from viral genomes that yield virions orvirus-like particles, which may or may not replicate independently asextrachromosomal elements. Virion particles containing the DNA for thehigh expression locus can be introduced into the host cells byinfection. The viral vector may become integrated into the cellulargenome. Examples of viral vectors for transformation of mammalian cellsare SV40 vectors, and vectors based on papillomavirus, adenovirus,Epstein-Barr virus, vaccinia virus, and retroviruses, such as Roussarcoma virus, or a mouse leukemia virus, such as Moloney murineleukemia virus. for mammalian cells, electroporation or viral-mediatedintroduction can be used. Further useful delivery systems and vehicleswe described herein (see, for example, section 2.3.1.).

Appropriate transformation transfection conditions can be determined bythose skilled in the art in view of the teachings herein.

Cells

The cells (e.g., host cells) employed in this invention include allmammalian cells, cell lines, and cell cultures. The cells can be derivedfrom mammals, such as mice, rats, or other rodents, or from primates,such as humans or monkeys. Mammalian germ cells or somatic cells can beemployed for this purpose. It will be understood that primary cellcultures or immortalized cells can be employed in carrying out thetechniques of this invention.

The mammalian cells are typically grown in cell culture fortransformation by the DNA. The cells can be fixed to a solid surface orgrown in suspension in appropriate nutrient media.

In the present invention, it is preferred that permanent (i.e., stable)transformation occurs. This is accompanied by integration of thetransforming DNA into the cellular genome by recombination. Insertionaltransformation, which results in the high expression locus being tagged,usually takes place by non-homologous recombination of the DNA constructcontaining the tag into a random genomic position, although it will beunderstood that homologous recombination can occur.

It will also be understood that no attempt has been made to determinewhether the antibiotic resistance gene and the second selectable markerintegrate in a single high expression locus in chromosomal DNA orwhether there are multiple sites of integration to form multiple highexpression loci in a given cell in any event, the mammalian cells ofthis invention contain at least one high expression locus.

The transformed cells obtained by the method of this invention can beemployed for the preparation of continuous cell lines in which the cellsare essentially immortal, or for the preparation of established celllines that have the potential to be subcultured in vitro. Continuouscell lines and established cell lines can be obtained from a variety oforganisms and organs, such as rodent embryos; primate kidneys; rodentand human tumors; and fibroblast, epithelial, or lymphoid cells. Cellsexhibiting the highest levels of expression can be cloned, if desired.

Examples of established cell lines that can be transformed by thetechniques of this invention are HeLa cells, CV-1 cells, CHO cells, 3T3cells, L cells, and TC7 cells. All of these cells are sensitive toaminoglyconide antibiotics, such as G418, and are capable of harboringkanamycin or neomycin resistance genes for expression therein.

Moreover, while this invention has been described with reference toexpression of a desired functional polypeptide, it will be understoodthat the polypeptide need not be the object of the invention. Thisinvention is useful for the production of eucaryotic gene transcriptionand expression products in general, including RNA.

This invention will be more fully described in the following Examples.

EXAMPLE 1 Constructs

pESN1dhfr was constructed as follows. PCR Primers D182E (M1) #1:5′-GGGTCACGACGAGATCATCGCCGT-3′ (SEQ ID NO:1) and D182 (M1) #2:3′-CGCATGCCCGACGGCGATGATCT-5′ (SEQ ID NO:2) were used to make pcDNA3M 1,which contains a mutated neo gene, from pcDNA3 (Invitrogen, Inc.), whichcontains wild-type neo. Throughout the application, where appropriate,it is noted where primers arc presented in 3′ to 5′ orientation. The PCRfragments were cloned into pCRII for TA cloning. The TA plasmidscontaining each separate mutation were digested with BssHII and SfuI toremove the region of the neo gene that contained the mutation at aminoacid residue 182 (Asp instead of wild-type Glu). pcDNA3M1, containingthe neo mutation at residue 182, was created by ligation of the M1 PCRfragment to pcDNA3 digested with BssHII and SfuI. The CMV IE promoterwas obtained by digesting pcMV6c with BalI/EcoRV, isolating the fragmentand ligating the fragment into pcDNA3M1 digested with the NruI andEcoRV. The resulting plasmid was terms pESN1. To add the Ad-DHFR gene tothe plasmid, pESN1 was blunt-ended digested with Bst1107I. The plasmidpmCSR (Chiron Corp.) was digested with XhoI and BamHI and the Ad-DHFRcassette isolated. The cassette was then treated with Klenow,blunt-ended and ligated into the blunt-cut pESN1 to create pESN1.dhfr.pcDNA3M2 was created by ligation of the M2 PCR fragment into pcDNA3digested with BssHII and SfuI. pcDNA3M3 was created by digestion ofpcDNA

pESN2dhfr was constructed as follows. Primers D261N (M2) #1:5′TCCCGCTCAGAAGAACTCGTTAAGAA-3′ (SEQ ID NO:3)and D261N (M2) #2:3′-CTATCGCCTTCTAACGAGTTCT-5′ (SEQ ID NO:4) were used to make pcDNA3M2,which contains a mutation in the neo gene at amino acid residue 261 (Asninstead of wild-type Asp). The PCR fragments were cloned into pCRII forTA cloning. The TA plasmids containing each separate mutation weredigested with BssHII and SfuI to remove the region of the neo gene thatcontained the mutation at amino acid residue 182 (Asp instead ofwild-type Glu). pcDNA3M2, containing the nco mutation at residue 261,was created by ligation of the M2 PCR fragment to pcDNA3 digested withBssHII and SfuI. The CMV IE promoter was obtained by digesting pcMV6cwith BalI/EcoRV, isolating the fragment and ligating the fragment intopcDNA3M2 digested with the NruI and EcoRV. The resulting plasmid wasterms pESN2. To add the Ad-DHFR gene to the plasmid, pESN2 wasblunt-ended digested with Bst1107I. The plasmid pmCSR (Chiron Corp.) wasdigested with XhoI and BamHI and the Ad-DHFR cassette isolated. Thecassette was then treated with Klenow, blunt-ended and ligated into theblunt-cut pENS1 to create pESN2.dhfr.

pESN3dhfr was constructed as follows. pcDNA3M3 was created by digestingpcDNA3M1 and pcDNA3M2 with SmaI and RsrII. The small fragment frompcDNA3M1 was then ligated to the large fragment of pcDNA3M2 to createpcDNA3M3, which contains mutations at residues 182 and 261 of the neogene. pESN3.dhfr was created as described above by inserting the CMV IEpromoter and DHFR genes.

pneo*dhfr*1 and pneo*dhfr*2 were created as follows. A murine DHFR genewas amplified from pSV2 using primers that create BstBI and StuI ends.The wild type Neor gene was removed from pcDNA3 and replaced with thewild-type murine DHFR gene to create pcDNA3dhfr. This plasmid was thenused in PCR to create mutant DHFR genes. For pneo*dhfr*l, two adjacentcodons in the 5′ region of dhfr (using pSV2dhfr) are replaced with 2stop codons thereby forming a 5′-mutated DHFR gene. For pneo*dhfr*2, twoadjacent codons in the 3′ region are replaced with stop codons. Themutations were introduced by PCR amplification of pcDNA3dhfr using theprimers shown in Table 1:

dhfr primer Sequence #53′-CTCGTTCTTGCCAATCCCCTATTATTGGGACACGGCGACGATGC-5′ (SEQ ID NO:5) #65′-AGGGAGGCTTTTTTGGAGGCCTAGGCT-3′ (SEQ ID NO:6) #75′-GCATCGTCGCCGTGTCCCAATAATAGGGGATTGGCAAGAACGGAG-3′ (SEQ ID NO:7) #83′-GGCATTCGAAGCATAGCTTTAGGAGGGGAGCAGAG-5′ (SEQ ID NO:8) #93′-CTCAGAGAGGACGCCTGGCTATTATGGGAGAAGTTTATATTTCCCC-5′ (SEQ ID NO:9) #105′-GGGGAAATATAAACTTCTCCCATAATAGCCAGGCGTCCTCTCTGAG-3′ (SEQ ID NO:10)

Mutations in the 5′ end of the dhfr gene were accomplished using primers#5, #6, #7 and #8. Mutations in the 3′ end were accomplished usingprimers #6, #8, #9 and #10 These new vectors were referred to aspXdhfr*1 and pXdhfr*2, where X refers to any transgene. Mutant forms ofthe neo resistance gene from pESN1, pESN2 or pESN3 are then insertedinto pdhfr*1 or pdhfr*2 to create pneo*dhfr*1 and pneo*dhfr*2. Theseconstructs can be used, as described below, to create a high expressionsite in a host cell.

pdhfr3′del was constructed as follows. pcDNA3DHFR3′ was digested withKpnI/EcoRV. Secreted alkaline protease (SeAP) was obtained by digestingpSeAP-Basic (Clontech) with ClaI, followed by Klenow treatment anddigestion with KpnI. The fragment was then ligated into pcDNA3DHFR3′under blunt/sticky conditions. pXdhfr3′del refers to this plasmidcontaining any transgene “X”.

pneo*dhfr5′del was constructed as follows. Plasmid pFC55 (Chiron Corp),which contains the same neo resistance gene from pcDNA3, was digestedwith BssHII and SfuI to insert the neo mutation fragments obtained asdescribed above. The BssHII/SfuI M1, M2 or M3 neo fragments were ligatedinto pFC55 to create pRSVM1neo, pRSVM2neo or pRSVM3neo, respectively.pCDNA3dhfr5′ (courtesy F. Randazzo) was digested with NruI/KpnI toremove the CMV promoter upstream of the polylinker region. pRSVM1neo wasdigested with NruI/KpnI and the resulting fragment inserted intopcDNA3dhfr5′, creating pRSVM1neodhfr5′. The resulting plasmid wasdigested with BssHII and KpnI to insert either RSVM2neo or RSVM3neofragments.

pCMVKm2: The pCMVKm2 vector was derived from pCMV6a (Chapman et al.,Nuc. Acids Res. (1991) 19:3979-3986) and comprises a kanamycinselectable marker, a ColEl origin of replication, a CMV promoterenhancer and Intron A, followed by an insertion site for the syntheticsequences described below followed by a polyadenylation signal derivedfrom bovine growth hormone—the pCMVKm2 vector differs from the pCMV-linkvector only in that a polylinker site was inserted into pCMVKm2 togenerate pCMV-link (polylinker at positions 1646 to 1697); pESN2dhfr andpCMVPLEdhfr, for expression in Chinese Hamster Ovary (CHO) cells; and,pAcC13, a shuttle vector for use in the Baculovirus expression system(pAcC13, was derived from pAcC12 which was described by Munemitsu S., etal., Mol Cell Biol. 10(1 1):5977-5982, 1990).

pCMV-link contains the CMV promoter (CMV IE ENH/PRO), bovine growthhormone terminator (BGH pA), kanamycin selectable marker (kan), and aColEI origin of replication (ColEI ori). A polycloning site is alsopresent following the CMV promoter sequences.

A restriction map for vectors pESNdhfr, pESN1dhfr, pESN2dhfr, pESN3dhfris presented in FIG. 2. In the figure, the CMV promoter (pCMV, hCMVIE),bovine growth hormone terminator (BGHpA), SV40 origin of replication(SV40Ori), neomycin selectable marker (Nco), SV40 polyA (SV4OpA),Adenovirus 2 late promoter (Ad2VLP), and the murine dhfr gene (mu dhfr)are indicated. A polycloning site is also indicated in the figurefollowing the CMV promoter sequences.

Briefly, construction of pCMVPLEdhfr was as follows. To construct a DHFRcassette, the EMCV IRES (internal ribosome entry site) leader wasPCR-amplified from pCite-4a+(Novagen, Inc., Milwaukee, Wis.) andinserted into pET-23d (Novagen, Inc., Milwaukee, Wis.) as an Xba-Ncofragment to give pET-EMCV. The dhfr gene was PCR-amplified frompESN2dhfr to give a product with a Gly-Gly-Gly-Ser spacer in place ofthe translation stop codon and inserted as an Nco-BamH1 fragment to givepET-E-DHFR. Next, the attenuated neo gene was PCR amplified from apSV2Neo (Clontech, Palo Alto, Calif.) derivative and inserted into theunique BamH1 site of pET-E-DHFR to give pET-E-DHFR/Neo(_(m2)). Finallythe bovine growth hormone terminator from pCDNA3 (Invitrogen, Inc.,Carlsbad, Calif.) was inserted downstream of the neo gene to givepET-E-DHFR/Neo(_(m2))BGHt. The EMCV-dhfr/neo selectable marker cassettefragment was prepared by cleavage of pET-E-DHFR/Neo(_(m2))BGHt.

The CMV enhancer/promoter plus Intron A was transferred from pCMV6a(Chapman et al., Nuc. Acids Res. (1991) 19:3979-3986) as a HindIII-Sa/lfragment into pUC 19 (New England Biolabs, Inc., Beverly, Mass.). Thevector backbone of pUC 19 was deleted from the Ndel to the Sap1 sites.The above described DHFR cassette was added to the construct such thatthe EMCV IRES followed the CMV promoter. The vector also contained anAmp¹ gene and an SV40 origin of replication.

pCMVKm2 vectors containing synthetic Gag expression cassettes have beendesignated as follows: pCMVKm2.GagMod.SF2, pCMVKm2.GagprotMod.SF2, andpCMVKm2.GagpolMod.SF2, as described in co-pending application AttyDocket Number 1621.002.

CAB constructs: All of the constructs described were made and clonedinto pAcC13 in the following manner. Since the CAB2 portion of allconstructs was generated by PCR all were sequenced confirmed by the ABIsystem of dye terminator sequence analysis. Initially all constructswere cloned into the intermediate cloning vectors PCRII by INVITROGEN orpBluescript SK+ by Stratagene. This was done for ease of sequencing andfurther subcloning.

All CAB2 constructs were generated in two parts. First the MCP portionof the CAB2 molecule from the KpnI site at the 5′ end to the HindIIIsite at amino acid 321 was generated and cloned into PCRII and analyzedto confirm correct sequence. This portion of CAB2 was then cloned intopBluescript SK+. Then into this SK+construct the remaining portion ofCAB2, from the HindIII site at amino acid 321 to the 5′ end EcoRI site,including the various 3′ end tails was cloned. The primers used togenerate the HindIII 5′ and 3′ ends were: CAB2H3-1CAGAAAGCTTTCTTCACATTTGTACGTTATTAC (SEQ ID NO:11) DAFTH5-1CAGAAAGCTTTGTGAAAATTCCTGGCGAGAAGGAC (SEQ ID NO:12)

The original HindIII to EcoRI PCR products containing the Short Heparinconsensus, the TFPI heparin consensus, and the GPI tail were initiallycloned into PCRII for sequence analysis. The original HindIII to EcoRIPCR products containing the Long Heparin consensus, the two fibronectintails, and the control sequence with no tail were cloned intopBluescript SK+for sequence analysis. After combining the two halves tomake the complete CAB2 in SK+for each of the variants the KpnI to EcoRIfragments were then subcloned into pAcCI3. All were expressed in insectcells using the Baculovirus system.

In addition to insect cell expression some of these constructs weregenerated for expression in mammalian systems. They include CAB2 withthe Long Heparin Consensus sequence, the TFPI Heparin binding tail, andthe control with no targeting sequence. All three of these CAB variantswere again generated with PCR in order to change the 5′ and 3′restriction sites required for cloning into mammalian system vectors.All variants were generated with 5′ end Not I and 3′ end Xba I sites andcloned into the intermediate vector PCRII for sequence confirmation. ThePCR primers used to generate Not I to Xba I clones were identical tothose used to generate the KpnI to EcoRI fragments except for the changein restriction site. Confirmed variants were then cloned into pGEM9Z(Promega). These were then transferred to a mammalian vector forexpression.

EXAMPLE 2 Identification and Correction of Aberrant mRNA Splicing

Cab2 is a chimeric, 110 kDa protein that combines features of membranecofactor protein (MCP) and decay accelerating factor (DAF) to inhibitcomplement activation. Higgins et al., supra. Initially, a CHO cell line(N2107) was established after transfection of a pcDNA3 vector(Invitrogen) carrying a sequence encoding CAB2.1. However, theproductivity of this cell line was low and, further, declined over time.

Analysis of the polypeptides present in the supernatant of the N2107cell line revealed a 42 kDa protein having unexplained CAB2.1-relatedsequence. Poly (A+) mRNA was isolated from cell pellets of cellstransiently transfected with CAB2-containing plasmids using Fast Track(InVitrogen). RT-PCR analysis was conducted as described in thePerkin-Elmer RT-PCR kit using primers which flank the coding region. Thepredominant PCR product encoded a predicted protein of 42 kDa. Alsopresent were several intermediate PCR products and a small amount offull-length (110 kDa) PCR product. Thus, RT-PCR revealed that the 42 kDacontaminant was caused by aberrant mRNA splicing between the chimericdomains of MCP and DAF in CAB2.1.

Aberrant splicing was corrected by overlapping PCR to producesplice-corrected sequences of CAB2.1, CAB4.2 and CAB4.3, by removingdonor and acceptor sites for RNA splicing which gave alternate proteinside processed product from mammalian cell in such a way as to preservethe amino acid so that it no longer resembled the consensus splicing.The changes were as follows:

Original Codons New Codons Donor Sites: amino acids 47-48 ATT GGT ATCGGG (Ile-Gly) aa 152 153 154 155 GGT AAG CCA GCA AAA CCC(Gly-Lys-Pro-Pro: SEQ ID NO:13) Acceptor Sites: amino acids 372 373 374TAT TTT CCA CTT ATT TCA AGC TCT (Tyr-Phe-Pro SEQ ID (SEQ ID NO:15)NO:14) aa 459 460 461 462 TAC TTC CCT CTA ATA AGC TCG AGT 463 464 (SEQID NO:17) (Leu Ile Ser Gly Ser Ser SEQ ID NO:16)

The changes were generated using overlapping PCR with the followingprimers. To change the Donor site starting at amino acid 47:

SACOL5P 5′-TATGGAGCTCATCGGGAAACCAAAA (SEQ ID NO:18)

SACOL3P 3′-GGGTTTTGGTTTCCCGATGAGCTC (SEQ ID NO:19)

To change the Donor site starting at amino acid 152:

NDE3P 3′-TTCACATATGGGGGGTTTTCCGCT (SEQ ID NO:20)

NDE5P 5′-AGCGGAAAACCCCCCATATGTGAA (SEQ ID NO:21)

To change the Acceptor site starting at amino acid 372:

ACCOL5P 5′-ATCACTCAGAATTACTTCCCTGTCGGT (SEQ ID NO:22)

ACCOL3P 3′-AACAGTACCGACAGGGAAGTAATTCTG (SEQ ID NO:23)

To Change the Acceptor site starting at amino acid 459:

ACCXH05P 5′-AATAAGCGGCTCGAGTGTCCAGTGG (SEQ ID NO:24)

ACCXH03P 3′-GACACTCGAGCCGCTTATTAGACAAAA (SEQ ID NO:25)

The 5′ Kpn primer used was:

MCPK5-2 5′-CAGAGGTACCATGGAGCCTCCCGGCCGCCGCGAG- (SEQ ID NO:26)

The 3′ XbaI primer used was;

DAFSXB32 3′-CAAATCTAGATTATCAACGGGTAGTACCTGAAGTGGTTC (SEQ ID NO:27)

Also used were the Hind III 5′ and 3′ primers named above.

Splice site deleted constructs included the complete CAB2, CAB2 with theTFPI Heparin binding tail, CAB2 with the partial and complete deletionof the Serine Threonine region to amino acid 570 and 561 respectively,and complete deletion of the serine threonine region with addition ofthe TFPI heparin consensus sequence.

The primers used for generating the Serine Threonine deletions at the 3′end of the molecule were:

STTDELE3: CAGAGAATTCTCATGTTGGTGGGACCTTGGA (SEQ ID NO:28)

STDELE31: CAGAGAATTCTCATTTTCCTCTGCATTCAGGAC (SEQ ID NO:29) CAB4-33P:

CAGAGAATTCTCACATATTTTTAACAAAAATTTCTTCATATGCTATTTTCACTCTCTGCTTCTTTCTTTTTCTTTTGGTTTTTTTTCCTCTGCATTCAGGTGGTGG (SEQ ID NO:30)

The constructs were made in two parts in the intermediate vectorpBluescript SK+as was done with the original CAB variants. All sequenceswere confirmed in SK+before completion of the final constructs inmammalian expression vectors. mRNA analyzed by RT-PCR and by Northernblotting showed no aberrant splicing and bands of the correct size.

EXAMPLE 3 Transfections

Splice-corrected CAB2.1 and CAB4.2 were transiently transfected intoCOS-7 cells using the LipofectAMINE reagent (BRL) for two hours. RT-PCRshowed only the correct full-length PCR product (110 kDa). Levels of CABprotein expressed in these cells are shown in Table 2.

TABLE 2 Expression Plasmid CAB 2 conc. (ng/mL) pESN3/CAB2.1C#8.2 733pESN3/CAB2.1C#8.2 614 pcDNA3/CAB2.1 <9.0

EXAMPLE 4 Transfection and Selection Methods

The following four transfection protocols of plasmids pESN1.dhfr.CAB2.1;pESN3.dhfr.CAB2.1, pESN1.dhrf.CAB42. or pESN3.dhfr.CAB4.2 wereconducted: (1) electroporation; (2) calcium phosphate (CaPo4) (LifeTechnologies, Gaithersburg, Md.); (3) LipofectAMINE (Life Technologies,Gaithersburg, Md.); and (4) TransIT LT-1 reagent (PanVera Corp.,Madison, Wis.). Transfections were conducted according to themanufacturer's instructions for all methods except electroporation.Approximately 3×10₆ cells were used per electroporation, and 50-80%confluent cells (5-8×10⁵ cells) for the three other transfectionmethods.

Electroporation was conducted as follows. CHO-DHFR⁻ cells weretyrpsinized with STV and media with 10% FBS was added to quench thereaction. Cells were washed twice with DMEM (with L-glutamine) andresuspended to approximately 6×10⁶ cells/mL. 500 μl of cells was addedto a sterile cuvette and between about 1-30 μg of DNA added. The cellswere electroporated at 330 volts and 975 μFarads capacitance. The cellswere plated into 15 mls of α-MEM/nonselective media (containing G418) ina 100 mm dish, incubated for 18-24 hours at 37° C. Media was thenreplaced with α-MEM selective medium for DHFR and the cells grown untilcolonies formed. Cells are replated (T75 flasks, 96 well plates or 100mm dish) and cultured in 20 nM methotrexate for 2-4 weeks at which pointthe concentration of methotrexate was raised to 40 nM methotrexate.

Approximately 10 μg of plasmid DNA was used per transfection.Transfected cells were COS (UCSF) or DG44 DHFR- CHO cells (Chiron Corp).After transfection, the cells were allowed to recover in non-selectivemedia (500 mL of non-selective media contains 431.5 mL of α-MEM, 50 mLFBS, non-dialyzed, 5 mL penicillin-streptomycin, 0.5 mL gentamicin, 10mL glutamine, 1 mL thymidine, 1 mL adenosine and 1 mL deoxyadenosine)for 1-2 days. The cells from one transfection were trypsinized asdescribed above, diluted into 20 mL of selective medium (500 mL ofselective media contains 434.5 α-MEM, 10 mL glutamine, 0.5 mLgentamicin, 5 mL penicillin-streptomycin, and 50 mls FBS non-dialyzed)and plated into one 96-well culture plate in appropriate selectionmedia, either 250 μg G418 (Gibco-BRL), α-MEM without nucleosides, 10%FBS (DHFR selection) or both media. Cells were maintained in theselective media for approximately 2-3 weeks, at which time untransfectedor transiently transfected cells were dead. Surviving colonies weretrypsinized and replaced into 24-well culture plates containing 1 mL ofselective media.

Growth-positive wells were tested for expression of CAB2.1 or CAB4.2using ELISA. CAB2.1 or CAB4.2 expressing mini-pools were scaled-up forDHFR selection and amplification with methotrexate (MTX). Resultsobtained indicated that CaPO4 was the most effective in obtaining stableexpression of the trans gene.

The expression of CAB2.1 or CAB4.2 in DHFR- CHO cells is shown in Table3.

TABLE 3 CAB2.1 CAB4.2 Construct (in pESN3dhfr) (in pESN3dhfr) No. oftransfections 8 3 No. wells screened 3360 480 No. selected in 24 wells/237 118 range of expression <0.50-4.10 μg/mL 0.035-5.50 μg/mL No.amplified in T75 flasks/ 11 19 range of expression 0.015-210 μg/mL0.494-2.33 μg/mL MTX amplification/ 1 2 range of expression 50.0-106.0μg/mL 20.0-75.0 μg/mL Selected for cloning 1 not determined

In addition to CAB2, expression of uPAR and VEGF-D has also beenachieved by calcium phosphate transfection of host cells with pESN2dhfrcontaining a transgene coding for uPAR or VEGF-D. Expression levelsbetween about 250 mg to about 1 mg/liter have been achieved.

Deposits of Strains Useful in Practicing the Invention

A deposit of biologically pure cultures of the following strains wasmade with the American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, Va. The accession number indicated was assignedafter successful viability testing, and the requisite fees were paid.The deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of viable cultures for a period ofthirty (30) years from the date of deposit and at least five (5) yearsafter the most recent request for the furnishing of a sample of thedeposit by the depository. The organisms will be made available by theATCC under the terms of the Budapest Treaty, which assures permanent andunrestricted availability of the cultures to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 U.S.C. §122 and the Commissioner's rules pursuant thereto(including 37 C.F.R. §1.12). Upon the granting of a patent, allrestrictions on the availability to the public of the deposited cultureswill be irrevocably removed.

These deposits are provided merely as convenience to those of skill inthe art, and are not an admission that a deposit is required under 35U.S.C. §112. The nucleic acid sequences of these plasmids, as well asthe amino acid sequences of the polypeptides encoded thereby, areincorporated herein by reference and are controlling in the event of anyconflict with the description herein. A license may be required to make,use, or sell the deposited materials, and no such license is herebygranted.

Plasmid, ATCC Accession Chiron Deposit Number Date Sent to ATCC Numberpneo3dhfr5′del, CMCC #5090 November 30, 1999 PTA-998 pneoldhfr5′del,CMCC #5089 November 30, 1999 PTA-1001 pESN2dhfr, CMCC #5086 November 30,1999 PTA-1002 pdfr3′del, CMCC #5093 November 30, 1999 PTA-1003pneo2dhfr5′del, CMCC #5088 November 30, 1999 PTA-1004 pESN1dhfr, CMCC#5085 November 30, 1999 PTA-1005 pESN3dhfr, CMCC #5087 November 30, 1999PTA-1006

The principles, preferred embodiments and modes of operation of thepresent invention have been described in the foregoing specification.The invention to be protected herein, however, is not to be construed aslimited to the particular forms disclosed, since these are to beregarded as illustrative rather than restrictive. Variations and changesmay be made by one of ordinary skill in the art without departing fromthe spirit of the invention.

32 1 24 DNA Artificial Sequence Description of Artificial Sequenceprimer D182E (M1) #1 1 gggtcacgac gagatcatcg ccgt 24 2 23 DNA ArtificialSequence Description of Artificial Sequence primer D182 (M1) #2 2tctagtagcg gcagcccgta cgc 23 3 26 DNA Artificial Sequence Description ofArtificial Sequence primer D261N (M2)#1 3 tcccgctcag aagaactcgt taagaa26 4 22 DNA Artificial Sequence Description of Artificial Sequenceprimer D261N (M2) #2 4 tcttgagcaa tcttccgcta tc 22 5 44 DNA ArtificialSequence Description of Artificial Sequence dhfr primer #5 5 cgtagcagcggcacagggtt attatcccct aaccgttctt gctc 44 6 27 DNA Artificial SequenceDescription of Artificial Sequence dhfr primer #6 6 agggaggcttttttggaggc ctaggct 27 7 45 DNA Artificial Sequence Description ofArtificial Sequence dhfr primer #7 7 gcatcgtcgc cgtgtcccaa taataggggattggcaagaa cggag 45 8 35 DNA Artificial Sequence Description ofArtificial Sequence dhfr primer #8 8 gagacgaggg gaggatttcg atacgaagcttacgg 35 9 46 DNA Artificial Sequence Description of Artificial Sequencedhfr primer #9 9 cccctttata tttgaagagg gtattatcgg tccgcaggag agactc 4610 46 DNA Artificial Sequence Description of Artificial Sequence dhfrprimer #10 10 ggggaaatat aaacttctcc cataatagcc aggcgtcctc tctgag 46 1133 DNA Artificial Sequence Description of Artificial Sequence CAB2H3-111 cagaaagctt tcttcacatt tgtacgttat tac 33 12 35 DNA Artificial SequenceDescription of Artificial Sequence DAFTH5-1 12 cagaaagctt tgtgaaaattcctggcgaga aggac 35 13 4 PRT CAB-2 13 Gly Lys Pro Pro 1 14 3 PRT CAB-214 Tyr Phe Pro 1 15 15 DNA Artificial Sequence Description of ArtificialSequence modified acceptor site 1 15 cttatttcaa gctct 15 16 6 PRT CAB-216 Leu Ile Ser Gly Ser Ser 1 5 17 15 DNA Artificial Sequence Descriptionof Artificial Sequence modified acceptor site 2 17 ctaataagct cgagt 1518 25 DNA Artificial Sequence Description of Artificial Sequence primerSACOL5P 18 tatggagctc atcgggaaac caaaa 25 19 24 DNA Artificial SequenceDescription of Artificial Sequence primer SACOL3P 19 ctcgagtagccctttggttt tggg 24 20 24 DNA Artificial Sequence Description ofArtificial Sequence primer NDE3P 20 tcgccttttg gggggtatac actt 24 21 24DNA Artificial Sequence Description of Artificial Sequence primer NDE5P21 agcggaaaac cccccatatg tgaa 24 22 27 DNA Artificial SequenceDescription of Artificial Sequence primer ACCOL5P 22 atcactcagaattacttccc tgtcggt 27 23 27 DNA Artificial Sequence Description ofArtificial Sequence primer ACCOL3P 23 gtcttaatga agggacagcc atgacaa 2724 25 DNA Artificial Sequence Description of Artificial Sequence primerACCXH05P 24 aataagcggc tcgagtgtcc agtgg 25 25 27 DNA Artificial SequenceDescription of Artificial Sequence primer ACCXH03P 25 aaaacagattattcgccgag ctcacag 27 26 34 DNA Artificial Sequence Description ofArtificial Sequence primer MCPK5-2 26 cagaggtacc atggagcctc ccggccgccgcgag 34 27 39 DNA Artificial Sequence Description of Artificial Sequenceprimer DAFSXB32 27 cttggtgaag tccatgatgg gcaactatta gatctaaac 39 28 31DNA Artificial Sequence Description of Artificial Sequence primerSTTDELE3 28 cagagaattc tcatgttggt gggaccttgg a 31 29 33 DNA ArtificialSequence Description of Artificial Sequence primer STDELE31 29cagagaattc tcattttcct ctgcattcag gac 33 30 106 DNA Artificial SequenceDescription of Artificial Sequence primer CAB4-33P 30 cagagaattctcacatattt ttaacaaaaa tttcttcata tgctattttc actctctgct 60 tctttctttttcttttggtt ttttttcctc tgcattcagg tggtgg 106 31 1848 DNA CAB2 CDS(7)..(1839) 31 ggccgc atg gag cct ccc ggc cgc cgc gag tgt ccc ttt ccttcc tgg 48 Met Glu Pro Pro Gly Arg Arg Glu Cys Pro Phe Pro Ser Trp 1 510 cgc ttt cct ggg ttg ctt ctg gcg gcc atg gtg ttg ctg ctg tac tcc 96Arg Phe Pro Gly Leu Leu Leu Ala Ala Met Val Leu Leu Leu Tyr Ser 15 20 2530 ttc tcc gat gcc tgt gag gag cca cca aca ttt gaa gct atg gag ctc 144Phe Ser Asp Ala Cys Glu Glu Pro Pro Thr Phe Glu Ala Met Glu Leu 35 40 45att ggt aaa cca aaa ccc tac tat gag att ggt gaa cga gta gat tat 192 IleGly Lys Pro Lys Pro Tyr Tyr Glu Ile Gly Glu Arg Val Asp Tyr 50 55 60 aagtgt aaa aaa gga tac ttc tat ata cct cct ctt gcc acc cat act 240 Lys CysLys Lys Gly Tyr Phe Tyr Ile Pro Pro Leu Ala Thr His Thr 65 70 75 att tgtgat cgg aat cat aca tgg cta cct gtc tca gat gac gcc tgt 288 Ile Cys AspArg Asn His Thr Trp Leu Pro Val Ser Asp Asp Ala Cys 80 85 90 tat aga gaaaca tgt cca tat ata cgg gat cct tta aat ggc caa gca 336 Tyr Arg Glu ThrCys Pro Tyr Ile Arg Asp Pro Leu Asn Gly Gln Ala 95 100 105 110 gtc cctgca aat ggg act tac gag ttt ggt tat cag atg cac ttt att 384 Val Pro AlaAsn Gly Thr Tyr Glu Phe Gly Tyr Gln Met His Phe Ile 115 120 125 tgt aatgag ggt tat tac tta att ggt gaa gaa att cta tat tgt gaa 432 Cys Asn GluGly Tyr Tyr Leu Ile Gly Glu Glu Ile Leu Tyr Cys Glu 130 135 140 ctt aaagga tca gta gca att tgg agc ggt aag ccc cca ata tgt gaa 480 Leu Lys GlySer Val Ala Ile Trp Ser Gly Lys Pro Pro Ile Cys Glu 145 150 155 aag gttttg tgt aca cca cct cca aaa ata aaa aat gga aaa cac acc 528 Lys Val LeuCys Thr Pro Pro Pro Lys Ile Lys Asn Gly Lys His Thr 160 165 170 ttt agtgaa gta gaa gta ttt gag tat ctt gat gca gta act tat agt 576 Phe Ser GluVal Glu Val Phe Glu Tyr Leu Asp Ala Val Thr Tyr Ser 175 180 185 190 tgtgat cct gca cct gga cca gat cca ttt tca ctt att gga gag agc 624 Cys AspPro Ala Pro Gly Pro Asp Pro Phe Ser Leu Ile Gly Glu Ser 195 200 205 acgatt tat tgt ggt gac aat tca gtg tgg agt cgt gct gct cca gag 672 Thr IleTyr Cys Gly Asp Asn Ser Val Trp Ser Arg Ala Ala Pro Glu 210 215 220 tgtaaa gtg gtc aaa tgt cga ttt cca gta gtc gaa aat gga aaa cag 720 Cys LysVal Val Lys Cys Arg Phe Pro Val Val Glu Asn Gly Lys Gln 225 230 235 atatca gga ttt gga aaa aaa ttt tac tac aaa gca aca gtt atg ttt 768 Ile SerGly Phe Gly Lys Lys Phe Tyr Tyr Lys Ala Thr Val Met Phe 240 245 250 gaatgc gat aag ggt ttt tac ctc gat ggc agc gac aca att gtc tgt 816 Glu CysAsp Lys Gly Phe Tyr Leu Asp Gly Ser Asp Thr Ile Val Cys 255 260 265 270gac agt aac agt act tgg gat ccc cca gtt cca aag tgt ctt aaa gtg 864 AspSer Asn Ser Thr Trp Asp Pro Pro Val Pro Lys Cys Leu Lys Val 275 280 285tcg act gac tgt ggc ctt ccc cca gat gta cct aat gcc cag cca gct 912 SerThr Asp Cys Gly Leu Pro Pro Asp Val Pro Asn Ala Gln Pro Ala 290 295 300ttg gaa ggc cgt aca agt ttt ccc gag gat act gta ata acg tac aaa 960 LeuGlu Gly Arg Thr Ser Phe Pro Glu Asp Thr Val Ile Thr Tyr Lys 305 310 315tgt gaa gaa agc ttt gtg aaa att cct ggc gag aag gac tca gtg atc 1008 CysGlu Glu Ser Phe Val Lys Ile Pro Gly Glu Lys Asp Ser Val Ile 320 325 330tgc ctt aag ggc agt caa tgg tca gat att gaa gag ttc tgc aat cgt 1056 CysLeu Lys Gly Ser Gln Trp Ser Asp Ile Glu Glu Phe Cys Asn Arg 335 340 345350 agc tgc gag gtg cca aca agg cta aat tct gca tcc ctc aaa cag cct 1104Ser Cys Glu Val Pro Thr Arg Leu Asn Ser Ala Ser Leu Lys Gln Pro 355 360365 tat atc act cag aat tat ttt cca gtc ggt act gtt gtg gaa tat gag 1152Tyr Ile Thr Gln Asn Tyr Phe Pro Val Gly Thr Val Val Glu Tyr Glu 370 375380 tgc cgt cca ggt tac aga aga gaa cct tct cta tca cca aaa cta act 1200Cys Arg Pro Gly Tyr Arg Arg Glu Pro Ser Leu Ser Pro Lys Leu Thr 385 390395 tgc ctt cag aat tta aaa tgg tcc aca gca gtc gaa ttt tgt aaa aag 1248Cys Leu Gln Asn Leu Lys Trp Ser Thr Ala Val Glu Phe Cys Lys Lys 400 405410 aaa tca tgc cct aat ccg gga gaa ata cga aat ggt cag att gat gta 1296Lys Ser Cys Pro Asn Pro Gly Glu Ile Arg Asn Gly Gln Ile Asp Val 415 420425 430 cca ggt ggc ata tta ttt ggt gca acc atc tcc ttc tca tgt aac aca1344 Pro Gly Gly Ile Leu Phe Gly Ala Thr Ile Ser Phe Ser Cys Asn Thr 435440 445 ggg tac aaa tta ttt ggc tcg act tct agt ttt tgt ctt att tca ggc1392 Gly Tyr Lys Leu Phe Gly Ser Thr Ser Ser Phe Cys Leu Ile Ser Gly 450455 460 agc tct gtc cag tgg agt gac ccg ttg cca gag tgc aga gaa att tat1440 Ser Ser Val Gln Trp Ser Asp Pro Leu Pro Glu Cys Arg Glu Ile Tyr 465470 475 tgt cca gca cca cca caa att gac aat gga ata att caa ggg gaa cgt1488 Cys Pro Ala Pro Pro Gln Ile Asp Asn Gly Ile Ile Gln Gly Glu Arg 480485 490 gac cat tat gga tat aga cag tct gta acg tat gca tgt aat aaa gga1536 Asp His Tyr Gly Tyr Arg Gln Ser Val Thr Tyr Ala Cys Asn Lys Gly 495500 505 510 ttc acc atg att gga gag cac tct att tat tgt act gtg aat aatgat 1584 Phe Thr Met Ile Gly Glu His Ser Ile Tyr Cys Thr Val Asn Asn Asp515 520 525 gaa gga gag tgg agt ggc cca cca cct gaa tgc aga gga aaa tctcta 1632 Glu Gly Glu Trp Ser Gly Pro Pro Pro Glu Cys Arg Gly Lys Ser Leu530 535 540 act tcc aag gtc cca cca aca gtt cag aaa cct acc aca gta aatgtt 1680 Thr Ser Lys Val Pro Pro Thr Val Gln Lys Pro Thr Thr Val Asn Val545 550 555 cca act aca gaa gtc tca cca act tct cag aaa acc acc aca aaaacc 1728 Pro Thr Thr Glu Val Ser Pro Thr Ser Gln Lys Thr Thr Thr Lys Thr560 565 570 acc aca cca aat gct caa gca aca cgg agt aca cct gtt tcc aggaca 1776 Thr Thr Pro Asn Ala Gln Ala Thr Arg Ser Thr Pro Val Ser Arg Thr575 580 585 590 acc aag cat ttt cat gaa aca acc cca aat aaa gga agt ggaacc act 1824 Thr Lys His Phe His Glu Thr Thr Pro Asn Lys Gly Ser Gly ThrThr 595 600 605 tca ggt act acc cgt tgatctaga 1848 Ser Gly Thr Thr Arg610 32 611 PRT CAB2 32 Met Glu Pro Pro Gly Arg Arg Glu Cys Pro Phe ProSer Trp Arg Phe 1 5 10 15 Pro Gly Leu Leu Leu Ala Ala Met Val Leu LeuLeu Tyr Ser Phe Ser 20 25 30 Asp Ala Cys Glu Glu Pro Pro Thr Phe Glu AlaMet Glu Leu Ile Gly 35 40 45 Lys Pro Lys Pro Tyr Tyr Glu Ile Gly Glu ArgVal Asp Tyr Lys Cys 50 55 60 Lys Lys Gly Tyr Phe Tyr Ile Pro Pro Leu AlaThr His Thr Ile Cys 65 70 75 80 Asp Arg Asn His Thr Trp Leu Pro Val SerAsp Asp Ala Cys Tyr Arg 85 90 95 Glu Thr Cys Pro Tyr Ile Arg Asp Pro LeuAsn Gly Gln Ala Val Pro 100 105 110 Ala Asn Gly Thr Tyr Glu Phe Gly TyrGln Met His Phe Ile Cys Asn 115 120 125 Glu Gly Tyr Tyr Leu Ile Gly GluGlu Ile Leu Tyr Cys Glu Leu Lys 130 135 140 Gly Ser Val Ala Ile Trp SerGly Lys Pro Pro Ile Cys Glu Lys Val 145 150 155 160 Leu Cys Thr Pro ProPro Lys Ile Lys Asn Gly Lys His Thr Phe Ser 165 170 175 Glu Val Glu ValPhe Glu Tyr Leu Asp Ala Val Thr Tyr Ser Cys Asp 180 185 190 Pro Ala ProGly Pro Asp Pro Phe Ser Leu Ile Gly Glu Ser Thr Ile 195 200 205 Tyr CysGly Asp Asn Ser Val Trp Ser Arg Ala Ala Pro Glu Cys Lys 210 215 220 ValVal Lys Cys Arg Phe Pro Val Val Glu Asn Gly Lys Gln Ile Ser 225 230 235240 Gly Phe Gly Lys Lys Phe Tyr Tyr Lys Ala Thr Val Met Phe Glu Cys 245250 255 Asp Lys Gly Phe Tyr Leu Asp Gly Ser Asp Thr Ile Val Cys Asp Ser260 265 270 Asn Ser Thr Trp Asp Pro Pro Val Pro Lys Cys Leu Lys Val SerThr 275 280 285 Asp Cys Gly Leu Pro Pro Asp Val Pro Asn Ala Gln Pro AlaLeu Glu 290 295 300 Gly Arg Thr Ser Phe Pro Glu Asp Thr Val Ile Thr TyrLys Cys Glu 305 310 315 320 Glu Ser Phe Val Lys Ile Pro Gly Glu Lys AspSer Val Ile Cys Leu 325 330 335 Lys Gly Ser Gln Trp Ser Asp Ile Glu GluPhe Cys Asn Arg Ser Cys 340 345 350 Glu Val Pro Thr Arg Leu Asn Ser AlaSer Leu Lys Gln Pro Tyr Ile 355 360 365 Thr Gln Asn Tyr Phe Pro Val GlyThr Val Val Glu Tyr Glu Cys Arg 370 375 380 Pro Gly Tyr Arg Arg Glu ProSer Leu Ser Pro Lys Leu Thr Cys Leu 385 390 395 400 Gln Asn Leu Lys TrpSer Thr Ala Val Glu Phe Cys Lys Lys Lys Ser 405 410 415 Cys Pro Asn ProGly Glu Ile Arg Asn Gly Gln Ile Asp Val Pro Gly 420 425 430 Gly Ile LeuPhe Gly Ala Thr Ile Ser Phe Ser Cys Asn Thr Gly Tyr 435 440 445 Lys LeuPhe Gly Ser Thr Ser Ser Phe Cys Leu Ile Ser Gly Ser Ser 450 455 460 ValGln Trp Ser Asp Pro Leu Pro Glu Cys Arg Glu Ile Tyr Cys Pro 465 470 475480 Ala Pro Pro Gln Ile Asp Asn Gly Ile Ile Gln Gly Glu Arg Asp His 485490 495 Tyr Gly Tyr Arg Gln Ser Val Thr Tyr Ala Cys Asn Lys Gly Phe Thr500 505 510 Met Ile Gly Glu His Ser Ile Tyr Cys Thr Val Asn Asn Asp GluGly 515 520 525 Glu Trp Ser Gly Pro Pro Pro Glu Cys Arg Gly Lys Ser LeuThr Ser 530 535 540 Lys Val Pro Pro Thr Val Gln Lys Pro Thr Thr Val AsnVal Pro Thr 545 550 555 560 Thr Glu Val Ser Pro Thr Ser Gln Lys Thr ThrThr Lys Thr Thr Thr 565 570 575 Pro Asn Ala Gln Ala Thr Arg Ser Thr ProVal Ser Arg Thr Thr Lys 580 585 590 His Phe His Glu Thr Thr Pro Asn LysGly Ser Gly Thr Thr Ser Gly 595 600 605 Thr Thr Arg 610

What is claimed is:
 1. An expression vector comprising: (a) a firstpolynucleotide encoding a first, crippled, selectable marker, whereinthe first, crippled selectable marker is selected from the groupconsisting of a sequence coding for a neomycin resistance gene producthaving a mutation at amino acid residue 182, a sequence coding for aneomycin resistance gene product having a mutation at amino acid residue261 and a sequence coding for a neomycin resistance gene product havingmutations at amino acid residues 182 and 261; (b) a secondpolynucleotide encoding a heterologous polypeptide of interest; and (c)a third polynucleotide encoding a second, amplifiable selectable marker.2. The expression vector of claim 1, wherein the third polynucleotideencodes for dihydrofolate reducatase (dhfr).
 3. The expression vector ofclaim 1, wherein the heterologous polypeptide of interest is selectedfrom the group consisting of complement activation blocker-2 (CAB-2),complement activation blocker-4 (CAB-4), urokinase-type plasminogenactivator receptor (uPAR), vascular endothelial growth factor-D (VEGF-D)and a viral protein.
 4. The expression vector of claim 1, wherein theheterologous polypeptide of interest is a viral glycoprotein.
 5. Amethod for producing a polypeptide of interest in a host cell,comprising (a) introducing an expression vector according to claim 1into suitable host cells; (b) selecting host cells which express thefirst and second selectable markers under conditions that select forstably integrated expression vectors; (c) growing the stably-transfectedhost cells under conditions which favor expression of the polypeptide ofinterest, and (d) isolating the polypeptide of interest.
 6. The methodof claim 5 wherein the polypeptide of interest is selected from thegroup consisting of CAB-2, CAB-4, uPAR, VEGF-D and a viral protein. 7.The method of claim 5, wherein the polypeptide is a viral protein. 8.The method of claim 5, wherein the host cell is a mammalian or insectcell.
 9. A host cell line that produces a polypeptide of interest,wherein said polypeptide is produced according to the method of claim 5.10. A plasmid selected from the group consisting of pESN1dhfr,pESN2dhfr, pESN3dhfr, pneo1dhfr5′del, pneo3dhfr5′del, pneo2dhfr5′del,and pdhfr3′del.
 11. A method for producing a polypeptide of interest ina host cell, comprising (a) introducing an plasmid according to claim 10into suitable host cells; (b) selecting host cells which express thefirst and second selectable markers under conditions that select forstably integrated plasmids; (c) growing the stably-transfected hostcells under conditions which favor expression of the polypeptide ofinterest, and (d) isolating the polypeptide of interest.
 12. A plasmiddesignated pESN1dhfr.
 13. A plasmid designated pESN2dhfr.
 14. A plasmiddesignated pESN3dhfr.
 15. A plasmid designated pneo1dhfr5′del.
 16. Aplasmid designated pneo3dhfr5′del.
 17. A plasmid designatedpneo2dhfr5′del.
 18. A plasmid designated pdhfr3′del.